Sequence analysis of two newly sequenced Archaea genomes, Methanococcus jannaschii and Methanobacterium thermoautotrophicum, fails to identify a recognizable homolog of existing cysteinyl-tRNA synthetases (CysRS). This raises the possibility that cysteine is charged by a novel pathway or by a novel cysteine tRNA synthetase in the methanogens. The goal of this proposed research is to investigate the mechanism of cysteinyl tRNA biosynthesis in M. jannaschii and M. thermoautotrophicum. Genes responsible for cysteine charging activities will be cloned by reverse genetics and genetic complementation. An overexpression system in E. coli will be used to produce sufficient material for biochemical characterizations. If a novel pathway is responsible for cysteine charging, the focus will be to characterize the pertinent enzymes and the reactions they catalyze. If direct charging exists, studies will focus on the comparison of the novel CysRS with its bacteria and eukaryotes relatives in terms of catalysis and substrate recognition. The unique RNA:protein interactions will also be studied by site-directed mutagenesis and attempts will be made to crystallize the proteins for future structural studies. The results should provide us insight into the evolution of the genetic code and the limits on the flexibility of the translational apparatus in prokaryotes and eukaryotes.
